Name:
2 points
Chem 465 Biochemistry II
Hour Exam 2
Multiple choice (4 points each):
1. Which of the following statements about the chemiosmotic theory is correct?
A) Electron transfer in mitochondria is accompanied by an asymmetric
release of protons on one side of the inner mitochondrial membrane.
B) It predicts that oxidative phosphorylation can occur even in the absence of an
intact inner mitochondrial membrane.
C) The effect of uncoupling reagents is a consequence of their ability to carry
electrons through membranes.
D) The membrane ATP synthase has no significant role in the chemiosmotic
theory.
E) All of the above are correct.
2. During oxidative phosphorylation, the proton motive force that is generated by
electron transport is used to:
A) create a pore in the inner mitochondrial membrane.
B) generate the substrates (ADP and Pi) for the ATP synthase.
C) induce a conformational change in the ATP synthase.
D) oxidize NADH to NAD+.
E) reduce O2 to H2O.
3. Which one of the following statements about human mitochondria is true?
A)About 900 mitochondrial proteins are encoded by nuclear genes.
B) Mitochondrial genes are inherited from both maternal and paternal sources.
C) rRNA and tRNA are imported from the cytoplasm and used in mitochondrial
protein synthesis.
D) The mitochondrial genome codes for all proteins found in mitochondria.
E) The mitochondrial genome is not subject to mutations.
4. Functional DNA is not found in:
A) bacterial nucleoids.
B) chloroplasts.
C) lysosomes.
D) mitochondria.
E) nuclei.
5. For a closed-circular DNA molecule of 10,000 base pairs in the fully relaxed form, the
linking number (Lk) is about:
A) 10,000.
B) 950.
C) 100.
D) 9.5.
E) 2.
6. The Meselson-Stahl experiment established that:
A)DNA polymerase has a crucial role in DNA synthesis.
B) DNA synthesis in E. coli proceeds by a conservative mechanism.
C) DNA synthesis in E. coli proceeds by a semiconservative mechanism.
D) DNA synthesis requires dATP, dCTP, dGTP, and dTTP.
E) newly synthesized DNA in E. coli has a different base composition than the
preexisting DNA.
7. E. coli DNA polymerase III:
A) can initiate replication without a primer.
B) is efficient at nick translation.
C) is the principal DNA polymerase in chromosomal DNA replication.
D) represents over 90% of the DNA polymerase activity in E. coli cells.
E) requires a free 5'-hydroxyl group as a primer.
Essay questions - Answer any 5.
1. In the respiratory chain there are 5 types of compounds used to transport electrons.
What are these five electron carriers, and how are they similar or different from each
other?
NADH Nicotinamide adenine dinucleotide. Nicotinic acid attached to an adenine
nucleotide. Freely water soluble - diffuses into and out of active sites of many water
soluble enzymes as a substrate so it can carry electrons from one enzyme to another.
Involved in 2 electron reactions. Generally pass electrons on to Flavoproteins
Flavoproteins - proteins that contain Flavin adenine dinucleotide. A flavin group
attached to a sugar sometimes attached to an adenine nucleotide. Either very tightly
bound to an enzyme or covalently bound to an enzyme so cannot transfer electrons
between different proteins. Proteins using FAD or FMN are called flavoproteins and
use the flavin as an electron carrying intermediate. Can participate in 1 or 2 electron
transfers. Flavoproteins generally pass their electrons on to Iron- Sulfur Proteins
Iron-Sulfur proteins - Proteins containing iron complexed with sulfur in iron-sulfur
clusters. Act as electron carrying intermediates within a complex, rather than as a
soluble electron carrier. Iron Sulfur proteins pass electron to Ubiquinone Q in Complex
I, Complex II and Fatty Acid oxidation pathways, and Iron sulfur proteisn accept
electrons from Ubiquinone Complex III and then pass the electrons on to cytochromes.
Ubiquinone (Q) a very nonpolar electron carrier. Not water soluble but freely soluble
within the lipid bilayer of a membrane, so it can diffuse into and out of active sites of
membrane bound proteins. Can carry one or two electrons
Cytochromes proteins with an iron bound to a heme group heme is tightly or covalently
bound to protein so, like the flavoproteins, it acts as an electron carrying intermediate in
an enzyme’s reaction, but does not carry electrons between proteins. The one
exception to this is cytochrome c which, as an entire protein, carries electrons from
complex III to complex IV
2. When you think about it, many of the enzymes involved in oxidative phosphorylation
are membrane bound enzymes that transport various ions across the mitochondrial
inner membrane. List every membrane bound enzyme in this process and the
chemicals that it transports into or across the membrane. In this list do not forget to
include the proteins that transport things like ATP and shuttle NADH equivalents across
the membrane as well.
Complex I - NADH:ubiquinine oxidoreductase - accepts electrons from NADH and
transfers them to ubiquinone (QH2) in the membrane, concomitantly removing 5 protons
from the inside of the mitochondira and pumping 4 of these protons to the outside of the
membrane.
Complex II - Succinate dehydrogenase - Oxidizes succinate to fumarate and transfers
electrons and protons to ubiqiunone (QH2) in the membrane
Complex III - Ubiquinone:cytochrome c oxidoreductase - Net effect is to transfer
electrons from QH2 in the membrane to two cytochrome c’s on the outside of the
membrane concomitantly transferring 2 protons from the inside of the mitochondira to
the outside as well releasing the two protons on the QH2 onthe outside as well.
Complex IV - cytochrome oxidase 2 electrons from 2 cytochrome c’s on the outside of
the mitochondrial membrane are combined with 2 protons on the inside of the
mitochondria to make 1 water molecules on the inside of the mitochondria. At the same
time 2 additional protons are pumped from the inside of the mito to the outside.
FoF1 (ATP synthase) synthesizes 1 ATP for every 4 protons that are transported into the
mitochondria from the outside.
Malate - á-ketoglutarate transporter Transports malate into and á-ketoglutarate out of
the mitochondira in the NADH shuttle system.
Glutamate - aspartate transporter Transports glutamate into and asparatate out of the
mitochonria in the NADH shuttle system.
Adenine nucleotide translocase - Transfers 1 ATP out and 1 ADP into the mitochondria
Phosphate translocase Transports 1 H2PO42- and one proton into the mitochondria.
Mitochondiral glycerol-3-phosphate dehydrogenase located on the outside of the
mitochondrial membrane, oxidizes glycerol-3-phosphate to dihydroxyacetone
phosphate and transfers the electrons and protons from this reaction to QH2 in the
membrane
The book never mentions it specifically but there must be a pyruvate translocase to
bring pyruvate into the mitochondirain
3. If only about 1.5% of the human genome actually codes for proteins, what the heck
does the other 98.5% code for? (List specific types of DNA, explain what the term
means, and give a rough % for how much of the genome is tied up in this kind of DNA.)
~ 28% is introns and non-coding regions
~ 45% is transpospons
~ 25 miscellaneous Includes 3% simple sequence repeats, large duplicates and stuff
cannot be cattagorized
4. All cccDNA from natural sources is negatively supercoiled. Does this mean that the
DNA is has a higher or a lower number of base pairs/ turn than the Watson Crick model
predicts? What advantages does negatively supercoiled DNA have over relaxed DNA?
Is it physically possible to make positively supercoiled DNA, and if so, how would you
do it?
Negatively supercoiled means that the DNA has less turns than it should, so there are
less turns in the DNA . If a given piece of DNA has less turns in it, there are more
base pairs per turn.
This is an advantage because it means that the DNA has already started to unwind, so
it is easier to bind proteins to it to further unwind it for replication.
Yes, it is physically possible to make positively supercoiled DNA. As far as ways to
make it, I just followed your answer and tried to see if it made sense.
I can think of at least two ways to make + supercoiled DNA.
This is the simple way - Positively supercoiled DNA has more turns in it than it
should. One way you can achieve this is to place the DNA in a solution with a high
ionic strength. This would interfere with the negative repulsion between the phosphates
in the backbone, and the DNA would twist up more tightly (less base pairs/turn). If one
added a topoisomerase at this point to relaxed the DNA and remove all the superhelical
twists, then removed the isomerase and returned the DNA to a low ionic strength
environment it would now have + supercoils.
Now for the more imaginative and complicated way. Remember the argument in the
book about how when DNA is binds to histones as the DNA wraps around the histone in
a negative sense, so that when you relax the DNA with a topoisomerase it now has
negative supercoils? If you synthesized histones with D amino acids instead of L amino
acids, the histone you make would be the mirror image. This mirror image histone
should wrap the DNA around it in the opposite sense , so when this should put +
supercoils into the DNA.
5. Direction - directions. Why can you have either 3'65' or 5'63' exonucleases, but you
can only have 5'63' polymerases? And how can a 5'63' polymerase synthesize DNA in
a bi-directional manner?
When we are degrading DNA and simply cutting phosphate bonds it doesn’t really
matter if you cut at a 3' or a 5' linkage, so exonucleases can cut from with end of the
DNA strand. Why you are making DNA, however, you are using nucleotides that only
have extra phosphates on the 5' end, so they can only be attached to the 3' end of a
DNA strand, so the DNA polymerase must work in the 5'63' direction.
Bidriectional synthesis is achieved by having the leading strand synthesized
continuously in the 5'63' direction, and by having short segments called Okazaki
fragments synthesized discontinuously on the lagging strand in the 5'63' direction then
getting stitched together using a ligase.
6. What are all the different enzymatic activities needed in a replisome and what are
their functions?
For initiation
You need proteins to recognize the origin site (dnaA).
You need to unwind the DNA (helicase)
You need to prime they polymerase with RNA synthesis (primase)
You need to remove torsional strain on the DNA as it unwinds (DNA gyrase)
You need to bind the exposed single stranded DNA (Single Strand Binding
Protein)
There are other proteins to make this all work, but I was interested in the main
enzymic activities
For elongation you need
You need to unwind the DNA (helicase - Dna B)
You need to prime they polymerase with RNA synthesis (primase)
You need to remove torsional strain on the DNA as it unwinds (DNA gyrase)
You need to bind the exposed single stranded DNA (Single Strand Binding
Protein)
Within the actual polymerase III you need some of the following activities
DNA polymerization to make the DNA
3'65' proofreading to check for errors
a clamp protein to clamp the polymerase onto the DNA
a clamp loading protein to put the clamp onto the DNA
In addition:
You need to removed the RNA primers (DNA pol I)
You need to seal the nicks between Okozaki fragments (ligase)
There are other proteins to make this all work, but I was interested in the main
enzymic activities
For termination you need
Tus protein - to recognize termination sequence
Topoisomerase IV - to separate the two DNA molecules that are twisted around
each other (catenanes)
Also needed are the SMC proteins to help keep the two new DNA molecules
separated from each other.
1. A
2. C
3. A
4. C
5. B
6. C
7. C